Spelling suggestions: "subject:"candida antarctic"" "subject:"bandida antarctic""
31 |
Exploiting enzyme promiscuity for rational designBranneby, Cecilia January 2005 (has links)
Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type. / QC 20100929
|
32 |
Surfactants based on natural products - enzymatic synthesis and functional characterizationViklund, Fredrik January 2003 (has links)
<p>Surfactants are molecules that contain a water-soluble and afat-soluble part. They have important functions in productssuch as detergents, cosmetics, pharmaceuticals and foods aswell as in many industrial processes. Surfactants are used onvery large scale, which makes it important to decrease theirimpact on the environment. This can be done by starting withnatural materials, by improving the synthetic methods and byreducing the use of limited resources such as energy andorganic solvents.</p><p>This thesis focuses on lipase-catalyzed synthesis ofsurfactants based on natural products. It also includesfunctional studies of the produced surfactants; as antioxidantsin oils, or as surfactants to solubilize pharmaceuticals.</p><p>Unsaturated fatty acid esters of ascorbic acid weresynthesized with catalysis by Candida antarctica lipase B in<i>t</i>-amyl alcohol and in ionic liquids. High yields ofascorbyl oleate were obtained in an ionic liquid that wasdesigned to improve the solubility of the fatty acid, when thereaction was performed under vacuum. Ascorbyl oleate wasamorphous and was a better antioxidant than ascorbyl palmitatein rapeseed oil.</p><p>Polyethylene glycol (PEG) stearate, PEG 12-hydroxystearateand a series of PEG 12-acyloxy-stearates were synthesized in avacuum-driven, solvent-free system using<i>C. antarctica</i>lipase B as catalyst. Critical micelleconcentration and solubilization capacity were determined forthe PEG 12-acyloxy-stearates. Their effects on living cellswere evaluated in studies of hemolysis and transepithelialelectrical resistance. Several PEG1500 12-acyloxy-stearateswere excellent solubilizers for pharmaceutical use and hadnegligible negative effects on living cells even at highconcentrations.</p><p>Enzymatic and chemo-enzymatic methods offer uniquepossibilities to synthesize surfactants of high purity. Pureand well-defined surfactants enable new applications and areimportant for the understanding of surfactantstructure-function relationships.</p>
|
33 |
Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase BSvedendahl, Maria January 2009 (has links)
Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).
|
34 |
Green Polymer Chemistry: Functionalization of Polymers Using Enzymatic CatalysisSen, Mustafa Yasin 15 December 2009 (has links)
No description available.
|
35 |
Enzyme Immobilization and Biocatalysis of PolysiloxanesPoojari, Yadagiri 13 April 2010 (has links)
No description available.
|
36 |
Strategies for cellulose fiber modificationPersson, Per January 2004 (has links)
This thesis describes strategies for and examples ofcellulose fiber modification.The ability of an engineered biocatalyst, acellulose-binding module fused to theCandida antarcticalipase B, to catalyze ring-openingpolymerization of e-caprolactone in close proximity tocellulose fiber surfaces was explored. The water content in thesystem was found to regulate the polymer molecular weight,whereas the temperature primarily influenced the reaction rate.The hydrophobicity of the cellulose sample increased as aresult of the presence of surface-deposited polyester. A two-step enzymatic method was also investigated. Here,Candida antarctica lipase B catalyzed the acylation ofxyloglucan oligosaccharides.The modified carbohydrates werethen incorporated into longer xyloglucan molecules through theaction of a xyloglucan endotransglycosylase. The modifiedxyloglucan chains were finally deposited on a cellulosesubstrate. The action ofCandida antarcticalipase B was further investigated inthe copolymerization of e-caprolactone and D,L-lactide.Copolymerizations with different e-caprolactone-to-D,L-lactideratios were carried out. Initially, the polymerization wasslowed by the presence of D,L-lactide. During this stage,D,L-lactide was consumed more rapidly than ε-caprolactoneand the incorporation occurred dimer-wise with regard to thelactic acid units. Morphological studies on wood fibers were conducted using asol-gel mineralization method. The replicas produced werestudied, without additional sample preparation, by electronmicroscopy and nitrogen adsorption. Information concerning thestructure and accessibility of the porous fiber wall wasobtained. Studies of never-dried kraft pulp casts revealedmicro-cavities and cellulose fibrils with mean widths of 4.7(±2) and 3.6 (±1) nm, respectively. Finally, cationic catalysis by simple carboxylic acids wasstudied. L-Lactic acid was shown to catalyze the ring-openingpolymerization of ε-caprolactone in bulk at 120 °C.The reaction was initiated with methylß-D-glucopyranoside, sucrose or raffinose, which resultedin carbohydrate-functionalized polyesters. The regioselectivityof the acylation was well in agreement with the correspondinglipase-catalyzed reaction. The polymerization was alsoinitiated with a hexahydroxy-functional compound, whichresulted in a dendrimer-like star polymer. The L-lactic acidwas readily recycled, which made consecutive reactions usingthe same catalyst possible. Keywords:Candida antarcticalipase B, cationic catalysis,cellulose-binding module, dendrimer, enzymatic polymerization,fiber modification, silica-cast replica, sol-gelmineralization, organocatalysis, xyloglucanendotransglycosylase
|
37 |
Rational redesign of Candida antarctica lipase BMagnusson, Anders January 2005 (has links)
This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity. Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze. Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis. The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C. Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.
|
38 |
Etude de l'acylation sélective de composés multifonctionnels par voie enzymatique : Application à la synthèse de pseudo-céramidesLe Joubioux, Florian 20 April 2012 (has links) (PDF)
Les céramides sont des lipides de la classe des sphingolipides issus de la N-acylation d'une base sphingoide par un acide gras. Ces lipides et leurs analogues suscitent un grand intérêt comme composants actifs dans les industries pharmaceutique et cosmétique. Parmi les biocatalyseurs capables de réaliser la synthèse de ce type de lipide, la lipase B de Candida antarctica semble être l'enzyme la plus adaptée à la production de " pseudo-céramides " à partir d'amino-polyols. Dans ce contexte, nous avons abordé l'étude de l'acylation de composés de type " amino-alcool "catalysée par la lipase B de Candida antarctica, en gardant à l'esprit une approche fondamentale afin d'élargir les connaissances actuelles sur ce sujet. La première partie de notre travail a ainsi traité de l'étude cinétique de l'acylation de composés monofonctionnels afin de déterminer les mécanismes réactionnels et l'énantio sélectivité de la lipase B de Candida antarctica pour les réactions de N-acylation et de O-acylation. Les parties suivantes de notre travail ont porté sur une étude structure-réactivité du substrat accepteur d'acyle et sur l'étude de l'influence du solvant utilisé (solvant organique ou liquide ionique) afin de déterminer les facteurs clés influençant la chimio sélectivité et la régio sélectivité de la lipase B de Candida antarctica lors de l'acylation de composés multifonctionnels de type " amino-alcool ". Finalement, à partir des connaissances acquises dans les différentes parties, nous avons développé et optimisé un procédé de synthèse enzymatique de " pseudo-céramides " (O,N-diacyl aminopropanediols) mis en oeuvre en réacteur continu à " lit fixe ".
|
39 |
Compréhension de l'énantiosélectivité de la lipase B de Candida antarctica : étude par modélisation moléculaire et expérimentationChaput, Ludovic 28 September 2012 (has links) (PDF)
La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l'obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l'industrie pharmaceutique. En effet, le principe actif de nombreux médicaments est efficace sous une forme énantio pure, l'autre forme chirale pouvant se révéler délétère pour l'organisme.Les travaux de la thèse s'intéressent à mieux comprendre l'origine de l'énantiosélectivité de la lipase B de Candida antarctica, en particulier pour la résolution d'alcools secondaires par des réactions de transestérification.Nous utilisons pour la première fois la méthode de la perturbation de l'énergie libre pour estimer la différence d'énergie libre entre les intermédiaires tétraédriques obtenus avec les formes R et S d'alcools énantiomères pour une série d'alcools secondaires, dans le but de prédire in silico l'énantiosélectivité de la CALB. Les paramètres cinétiques apparents d'une réaction avec deux alcools substrats énantiopurs sont expérimentalement déterminés et permettent de définir la contribution respective du Km et du kcat de chaque énantiomère pour la définition de l'énantiosélectivité. L'étude expérimentale de l'effet d'empreinte par des molécules co-substrats est réalisée,ainsi qu'une étude par modélisation moléculaire de l'effet d'empreinte par le premier ester substrat de la réaction qui pourrait modifier la conformation du site actif de la CALB. La troisième partie porte sur l'étude de la CALB et de trois variants (T42V, S47A et T42V/S47A) chez lesquels les acides aminés dans la poche stéréospécifiques ont mutés. T42V et S47A permettent d'obtenir une augmentation de l'énantiosélectivité. L'étude propose une étude détaillée de la conformation du site actif à partir de simulations de trajectoires de dynamique moléculaire
|
40 |
Strategies for cellulose fiber modificationPersson, Per January 2004 (has links)
<p>This thesis describes strategies for and examples ofcellulose fiber modification.The ability of an engineered biocatalyst, acellulose-binding module fused to the<i>Candida antarctica</i>lipase B, to catalyze ring-openingpolymerization of e-caprolactone in close proximity tocellulose fiber surfaces was explored. The water content in thesystem was found to regulate the polymer molecular weight,whereas the temperature primarily influenced the reaction rate.The hydrophobicity of the cellulose sample increased as aresult of the presence of surface-deposited polyester.</p><p>A two-step enzymatic method was also investigated. Here,Candida antarctica lipase B catalyzed the acylation ofxyloglucan oligosaccharides.The modified carbohydrates werethen incorporated into longer xyloglucan molecules through theaction of a xyloglucan endotransglycosylase. The modifiedxyloglucan chains were finally deposited on a cellulosesubstrate.</p><p>The action of<i>Candida antarctica</i>lipase B was further investigated inthe copolymerization of e-caprolactone and D,L-lactide.Copolymerizations with different e-caprolactone-to-D,L-lactideratios were carried out. Initially, the polymerization wasslowed by the presence of D,L-lactide. During this stage,D,L-lactide was consumed more rapidly than ε-caprolactoneand the incorporation occurred dimer-wise with regard to thelactic acid units.</p><p>Morphological studies on wood fibers were conducted using asol-gel mineralization method. The replicas produced werestudied, without additional sample preparation, by electronmicroscopy and nitrogen adsorption. Information concerning thestructure and accessibility of the porous fiber wall wasobtained. Studies of never-dried kraft pulp casts revealedmicro-cavities and cellulose fibrils with mean widths of 4.7(±2) and 3.6 (±1) nm, respectively.</p><p>Finally, cationic catalysis by simple carboxylic acids wasstudied. L-Lactic acid was shown to catalyze the ring-openingpolymerization of ε-caprolactone in bulk at 120 °C.The reaction was initiated with methylß-D-glucopyranoside, sucrose or raffinose, which resultedin carbohydrate-functionalized polyesters. The regioselectivityof the acylation was well in agreement with the correspondinglipase-catalyzed reaction. The polymerization was alsoinitiated with a hexahydroxy-functional compound, whichresulted in a dendrimer-like star polymer. The L-lactic acidwas readily recycled, which made consecutive reactions usingthe same catalyst possible.</p><p><b>Keywords:</b><i>Candida antarctica</i>lipase B, cationic catalysis,cellulose-binding module, dendrimer, enzymatic polymerization,fiber modification, silica-cast replica, sol-gelmineralization, organocatalysis, xyloglucanendotransglycosylase</p>
|
Page generated in 0.0528 seconds